Project Summary/Abstract Abilita Bio (AB), an innovation driven biotechnology company, requests SBIR Phase I funding for the discovery of novel therapeutic antibodies targeting G protein-coupled receptors (GPCRs) for the treatment of metastatic breast cancer. In cancer therapy each treatment modality has its own limitations. Clinical studies clearly indicate that immunotherapy with checkpoint inhibitors (PD-1/PD-L1, CTLA-4 etc.) can achieve a persistent immune response with improved clinical outcomes. However, this approach is especially effective when administered in combination therapy with targeted therapy aiming at essential components of tumor spreading, homing and growth especially in metastatic cancer. Metastasis is the major factor limiting survival in cancer patients. Therefore, interfering with the molecular mechanisms controlling the metastatic behavior of tumor cells is essential for successfully treating cancer. Experimental and clinical evidence supports the notion that one of the most important mechanisms operating in metastasis involves homeostatic chemokines and their receptors. Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. Tumor cell migration from the primary tumor site to the metastatic homing sites shares many similarities with leukocyte trafficking, which is critically regulated by chemokine-binding GPCRs. Interestingly, several studies have established that prostaglandin E2 (PGE2) secretion by breast cancer cells can upregulate CCR7 expression via activation of prostaglandin-binding GPCRs, EP2 and EP4. This proposal focuses on targeting key GPCRs critically involved in tumor cells metastatic migration and organ-specific invasion using therapeutic antibodies. The use of therapeutic antibodies to target GPCRs has a tremendous potential for the treatment of cancer because of their high affinity, exquisite specificity and low toxicity relative to small molecules. However, GPCRs therapeutic antibody discovery is extremely challenging because of their inherent low stability, low expression and conformational heterogeneity outside their natural membrane environment. In fact, only 1 GPCR-targeting antibody has been approved to date, and only in Japan. In order to overcome the afore mentioned barriers, Abilita Bio offers an innovative solution through the generation of Enabled Membrane Proteins (EMPs?), representing structurally and functionally enhanced versions of natural receptors with significant improved expression and thermostability. The availability of highly expressing, stable and functionally folded EMPs will enable the isolation of novel therapeutic antibodies using in vitro antibody display methods. Funds are requested to (a) generate mutational gene libraries for proposed targets and select for enhanced variants using our directed evolution system; (b) characterize variants for expression, function and enhanced stability in mammalian cells; (c) develop biopanning assay tools using evolved EMPs as antigens and screen phage display antibody libraries for the isolation of preliminary antibody leads to be optimized and developed in Phase II.